Calculating rabies virus neutralizing antibodies titres by flow cytometry

• Main Authors: BORDIGNON Juliano, COMIN Fabiano, FERREIRA Sílvia Córdoba P., CAPORALE Graciane M. M., LIMA FILHO José Hermênio Cavalcante, ZANETTI Carlos Roberto
• Format: Article in Journal/Newspaper
• Language: English
• Published: Universidade de São Paulo (USP) 2002
• Subjects: Rabies virus; Flow cytometry; Neutralizing antibodies; Arctic medicine. Tropical medicine; RC955-962; Infectious and parasitic diseases; RC109-216; Arctic
• Online Access: https://doaj.org/article/e2a58b99cc4b42b8b1d940213ab36c31

The determination of the rabies neutralizing antibody (VNA) response after immunization against rabies is an acceptable index of the efficacy of a vaccine and a successful treatment. Several tests have been developed in attempt to improve the assessment of VNA, from mice inoculation to cell-culture fluorescence inhibition tests. All of them, however, present special difficulties in terms of reading or accuracy. The present study describes a neutralization test performed in cell-culture appraised by flow cytometry (FC). Serial dilutions of the serum samples were mixed in vitro with rabies virus before the addition of BHK-21 cells. After 24h-incubation, cells were released by trypsin treatment, fixed and permeabilized with a p-formaldehyde solution and stained with a rabies virus nucleocapsid protein-specific antibody conjugate. The percentage of virus infection inhibition caused by specific antibodies present in the serum were evaluated in a Beckton & Dickinson FACSCalibur® flow cytometer. A correlation curve between the IU/ml content and the percentage of infective inhibition was built with a reference serum and the VNA titers of serum samples were obtained by extrapolation. Titers obtained by FC and standard test showed an effective pairing results (p < 0.01), with a correlation coefficient (r) = 0.7. These results permit to envisage the FC as a suitable technique to evaluate VNA in sera from immunized animals and likely in human serum samples. Nevertheless, new studies comparing FC to gold-standard techniques are required for determining the FC values of Sensibility and Specificity .